ABSTRACT M. tuberculosis (Mtb) is a highly successful human pathogen that remains one of the world's major causes of illness and death. The emergence of multi and extensively drug resistant strains of Mtb raises concern about untreatable tuberculosis (TB) and demands the development of unconventional therapies to shorten the TB treatment and to effectively eradicate subpopulation of the drug-tolerant bacteria. Mtb takes several days to die following incubation with lethal concentrations of compounds in vitro and in vivo. Our laboratory have identified the global metabolic remodeling of Mtb during exposure to each class of available anti-TB drugs and discovered ?escape? pathways and enzymes, simultaneously belonging to number of diverse metabolic pathways, allowing the pathogen to shift the transcriptional response and survive longer, even in presence of bactericidal compounds. One of the synergistic targets identified in our study is the LpqY-SugABC transporter system involved in the uptake and recycling of the disaccharide trehalose, a constituent of the mycobacterial cell wall. The transporter genes of LpqY system are non-essential for Mtb growth in vitro, however, they are vital for survival in nutrient-deprived microenvironment of host cells and for growth in vivo. Our preliminary data shows that the LpqY system can serve as the promising drug-target that has a synergistic effect with existing anti-TB drugs, killing Mtb faster in vitro and in macrophages. The overall goal of this project is to identify antimicrobials that inhibit the function of LpqY-SugABC. While the Mtb knockout clone of the LpqY- SugABC fails to internalize and process the extracellular trehalose in vitro and in infected macrophages, using the complemented clone, we can demonstrate that the specific incorporation of trehalose probe occurs via the LpqY system in vitro as well as during infection of THP-1 macrophages. Therefore, in the Aim 1a, we will achieve the high-throughput phenotypic screening by utilizing the fluorescein-labeled trehalose, and will identify active compounds that can block metabolic labeling of Mtb and, consequently, the function of the LpqY transporter system directly within infected human macrophages. By utilizing the tomato red protein- expressing Mtb strain in the Aim 1a, we will simultaneously distinguish if identified compounds have an inhibitory effect on intracellular Mtb growth as well. The Aim 1b will establish the killing kinetics of identified compounds in vitro, and will test efficacy and synergy with frontline anti-TB drugs against Mtb during infection of macrophages. In the Aim 1c, we will determine which component of the LpqY-SugABC system is targeted by each compound identified in this study. The proposal uses the rationally discovered target that contributes to intrinsic resistance and drug-tolerant phenotype of Mtb promoting bacterial survival within host macrophages. Equally significant factor is that inactivation of the LpqY system synergistically accelerates Mtb killing by current anti-TB compounds and, thus, the proposed screening has the potential to lead to novel drugs that can shorter therapy time and decrease chances for development of the drug resistance and persistence mechanisms.